The retinal pigment epithelium (RPE) is essential for development and function of the eye as it mediates photoreceptor outer segment renewal, regeneration of visual pigments, trans-epithelial transport, retinoid storage and protection against oxidative damage. Accordingly, alterations in RPE structure or physiology caused by environmental or genetic perturbations can ultimately cause blindness. Importantly, improper RPE development impairs eye growth and can result in severe congenital defects such as microphthalmia. Thus, it is critical to identify the mechanisms underlying normal development of the RPE. While some genes crucial for RPE development and function (e.g. Mitf, Otx2) are known, it is unclear how RPE-specific gene expression is initiated and maintained. Our preliminary data in mouse optic vesicle explants suggests that the surrounding extraocular mesenchyme produces a signal(s) to promote RPE development. Furthermore, we have evidence that the Wnt/[unreadable]-catenin pathway is essential for continuation of embryonic RPE differentiation. We hypothesize that activin receptor activation by the mesenchyme acts as an early signal to induce the RPE, while Wnt/[unreadable]-catenin signaling acts later to ensure RPE maintenance and function. In this project, we propose to examine in mouse the exact temporal requirement of extraocular mesenchyme and its role in activating the activin and Wnt/[unreadable]-catenin pathways using explant cultures and tissue-specific gene disruption (Aim 1). To investigate the role of Wnt/[unreadable]-catenin signaling in maintenance of the peri- and postnatal RPE, we will perform inducible, tissue-specific inactivation of -catenin. We will also determine whether Wnt/[unreadable]-catenin through TCF/LEF transcription factors directly activates RPE-specific gene expression using luciferase and ChIP assays (Aim 2). Using AP2a gene disruption in the mouse embryo and FGF treatment of optic vesicle explants, we propose to test whether ectopic and sustained activation of the Wnt/[unreadable]-catenin pathway is sufficient to block transdifferentiation of RPE into retina (Aim 3). Together, these experiments will advance our understanding of the signals that control RPE differentiation during mammalian eye development and may provide clues for therapeutic treatment of degenerative diseases in the eye. PUBLIC HEALTH RELEVANCE: The retinal pigment epithelium (RPE) is essential for development and function of the vertebrate eye. Abnormal RPE development impairs eye growth and results in congenital defects such as microphthalmia. Signaling between tissues and cells is a fundamental mechanism by which development is regulated;however, many of the regulatory signals controlling RPE development remain to be defined. Together, the goal of experiments proposed here will advance our understanding of the signals that control RPE development and may provide clues for new putative targets for therapeutic treatment of degenerative diseases in the eye.